Methodology for the efficient generation of fluorescently tagged vaccinia virus proteins

J Vis Exp. 2014 Jan 17:(83):e51151. doi: 10.3791/51151.

Abstract

Tagging of viral proteins with fluorescent proteins has proven an indispensable approach to furthering our understanding of virus-host interactions. Vaccinia virus (VACV), the live vaccine used in the eradication of smallpox, is particularly amenable to fluorescent live-cell microscopy owing to its large virion size and the ease with which it can be engineered at the genome level. We report here an optimized protocol for generating recombinant viruses. The minimal requirements for targeted homologous recombination during vaccinia replication were determined, which allows the simplification of construct generation. This enabled the alliance of transient dominant selection (TDS) with a fluorescent reporter and metabolic selection to provide a rapid and modular approach to fluorescently label viral proteins. By streamlining the generation of fluorescent recombinant viruses, we are able to facilitate downstream applications such as advanced imaging analysis of many aspects of the virus-host interplay that occurs during virus replication.

Publication types

  • Research Support, Non-U.S. Gov't
  • Video-Audio Media

MeSH terms

  • Green Fluorescent Proteins / biosynthesis*
  • Green Fluorescent Proteins / genetics
  • Recombinant Fusion Proteins / biosynthesis*
  • Recombinant Fusion Proteins / genetics
  • Vaccinia virus / genetics*
  • Vaccinia virus / metabolism*
  • Viral Proteins / biosynthesis*
  • Viral Proteins / genetics

Substances

  • Recombinant Fusion Proteins
  • Viral Proteins
  • Green Fluorescent Proteins